Pharmaceutical preparation containing magnetic vesicular particles, manufacturing method thereof and diagnostic therapeutic system

ABSTRACT

A pharmaceutical preparation containing magnetic vesicular particles is disclosed, wherein the magnetic vesicular particles each include magnetic microparticles within a lipid membrane, an organic compound having at least two groups selected from the group consisting of a hydroxyl group, a carboxyl group, a carbamoyl group, an amino group, a mercapto group, a sulfo group, a dithio group, a thiocarboxyl group and a dithiocarboxyl group is bonded to the magnetic microparticle, and the magnetic vesicular particles satisfy the following equation:
 
0.05≦ R /( r ×100)≦1.5
 
wherein R represents an average grain size of the magnetic vesicular particles and r represents an average particle size of magnetic microparticles included in the magnetic vesicular particles.

This application is a Divisional of U.S. patent application Ser. No.11/268,213 filed Nov. 7, 2005, pending, which, in turn, claimed thepriority from Japanese Patent Application No. JP2004-326431, filed onNov. 10, 2004, both of which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to pharmaceutical preparations containingmagnetic vesicular particles in which magnetic microparticles are bondedwith an organic compound, and a manufacturing method thereof, and adiagnostic therapeutic system.

BACKGROUND OF THE INVENTION

Development of “magnetic nano-beads” has been promoted as one ofpromising medical materials for use in next-generation medicaltechniques. The magnetic nano-beads (hereinafter, also denoted asmagnetic microparticles) are nano-level size microparticles of ferrite(solid solution of Fe₃O₄ and γ-Fe₂O₃). Recently, there has beendeveloped a technique of synthesizing them under mild conditions of 4 to25° C. in the range of a neutral pH, as described in JP-A No.2002-128523 (hereinafter, the term, JP-A refers to Japanese PatentApplication Publication). Magnetic nano-beads for medical use have beenproposed, in which drugs or physiologically active materials are allowedto be fixed onto (attached to or included in) the magneticmicroparticles covered with dextran, lipid, liposome, polymer or thelike, as described in JP-A Nos. 2002-128523 and 9-110722. As applicationof such magnetic microparticles in the medical field are proposedapplications to contrast medium material used for MRI diagnosis or amedicine-transporting carrier, and thermotherapy employing heatgeneration due to hysteresis loss of magnetic microparticles in the highfrequency magnetic field; there was further proposed concurrentperformance of diagnosis and therapy of cancer by the combination ofboth of the foregoing, as described in “BIO INDUSTRY” vol. 21, No. 8,page 48-54 (2004).

Thermotherapy for cancer (Hyperthermia) has been proposed for severaldecades and is one of the cancer therapies studied. The principle oftherapy employs a property of cancer cells being weaker to heat thannormal cells, thereby giving therapy with artificially maintaining ahigh temperature environment. Thermotherapy for cancer is a non-invasivetreatment as compared to surgical removal operation which is generallyconducted for cancer therapy. Comparing to chemotherapy or radiotherapywhich often results in adverse effects, it is selective and its adverseeffect becomes lower. Thus, it is a treatment which makes preservationof organs feasible and enhances the patient's quality of life. It istherefore a treatment suitable for an early cancer, or aged persons orinfants who are intolerable to operative invasion or adverse effects.Conventional thermotherapy has employed techniques such as inductionheating or focused ultrasonic heating and a recent alternate magneticfield heating using a ferrite type MRI contrast medium, and each ofthese therapies has merits and demerits. Accordingly, there is studiedthe possibility of using the above-described ferrite type particles as anovel heat-generating element in the alternating magnetic field, asdescribed in “BIO INDUSTRY” vol. 21, No. 8, page 48-54 (2004).

Feasibility of putting employment of magnetic microparticles intopractice in the medical field relies on capability of maintaining aphysiologically active material, a treatment drug or the like within thebeads or capability of its selective delivery to an intended site(targeting capability). In this regard, the magnetic vesicular particleswhich have been proposed so far, still leave room for improvement. Inenclosure of magnetic microparticles within liposome vesicles, variousproblems relating preparation of the liposome, for example, residue oforganic solvents and stability of a liposome structure retard itspractical use.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a pharmaceuticalpreparation containing magnetic vesicular particles which enablesselective delivery of agents to a tumor site (targeting capability), isapplicable to a contrast medium material or drug transport carrier andis also usable in thermotherapy employing heat-generation, and diagnosisand treatment of cancer by the combination thereof, and a manufacturingmethod thereof and a diagnostic therapeutic system by use thereof.

One aspect of the invention is directed to a pharmaceutical preparationcomprising magnetic vesicular particles, wherein the magnetic vesicularparticles each include one or more magnetic microparticles within alipid membrane, the magnetic microparticles are each bonded to anorganic compound having in its molecule at least two bonding groupsselected from a hydroxyl group, a carboxyl group, a carbamoyl group, anamino group, a mercapto group, a sulfo group, a dithio group, athiocarboxyl group and a dithiocarboxyl group, and the magneticvesicular particles satisfy the following equation:0.05≦R/(r×100)≦1.5wherein R represents an average grain size of the magnetic vesicularparticles and r represents an average particle size of the wholemagnetic microparticles included in the magnetic vesicular particles.

The magnetic vesicular particles preferably satisfy the followingequation:0.05≦R/(r×100)≦1.0

It is preferable that the magnetic microparticles are formed at a pH of7 to 10 and a temperature of 3 to 30° C. in the presence of an organiccompound having at least two groups selected from a hydroxyl group, acarboxyl group, a carbamoyl group, an amino group, a mercapto group, asulfo group, a dithio group, a thiocarboxyl group and a dithiocarboxylgroup.

It is preferable that the average particle size (r) of the magneticmicroparticles be within the range of 1 nm to 30 nm and the maincomponent of the magnetic microparticles be a ferrite.

It is preferable that the magnetic vesicular particles which include oneor more magnetic microparticles within a lipid membrane are liposomeparticles (or vesicles), and the surface electric charge of the liposomeis positive.

The preparation containing cover magnetic grains are used preferably asan imaging agent and/or a therapeutic agent for tumor.

In the preparation containing magnetic vesicular particles, aphysiologically active material and/or an antitumor-active material arebonded directly or via a linkage substance to the surface of the lipidmembrane. The imaging agent is used preferably as a contrast medium foruse in an ultrasonic imaging diagnostics, a nuclear magnetic resonanceimaging diagnostics or an X-ray imaging diagnostics.

When the preparation containing magnetic vesicular particles is used asan imaging agent (or a contrast medium) in an ultrasonic imagingdiagnosis apparatus, a nuclear magnetic resonance imaging diagnosisapparatus or a radiographic image diagnosis apparatus, performing scanswithin not less than 1 min. and not more than 48 hr. after thepreparation containing magnetic vesicular particles is dosed into thevein of an examinee, can result in enhanced capability to detecttumorous tissue.

On the other hand, when the preparation containing magnetic vesicularparticles is injected into the region near tumorous tissue of anexaminee, enhanced capability of detecting tumorous tissue can beachieved by performing scan within not less than 0.5 min. and not morethan 36 hr. after the start of injection, using an ultrasonic imagingdiagnostic apparatus, a nuclear magnetic resonance imaging diagnosticapparatus or a radiographic imaging diagnostic apparatus.

In the preparation containing magnetic vesicular particles, preferably,at least one selected from a physiologically functional material,additively stabilizing material, medicinally active material,medicinally active chelating material, antitumor-active material,immunopotentiating material, cell fusion material, and gene transfermediating material is bonded directly or via a linking material to theoutermost layer of the lipid membrane.

The above-described therapeutic agent is preferably an agent used forthermotherapy and the thermotherapy is performed, while being subjectedto exposure to energy. The exposure to energy enables raising thetemperature of the tumorous tissue in the close vicinity of the magneticvesicular particles. The exposure to energy is preferably exposure to analternating magnetic field or exposure to ultrasonic waves and of these,exposure to an alternating magnetic field at a frequency of 25 to 500kHz is specifically preferred.

When the preparation containing magnetic vesicular particles is used asa therapeutic agent, exposing an examinee to an alternating magneticfield or ultrasonic waves within not less than 1 min. and not more than48 hr. after the preparation containing magnetic vesicular particles isdosed into the vein of the examinee, the temperature of a tumoroustissue can be raised in the close vicinity of the magnetic vesicularparticles.

On the other hand, when the preparation containing magnetic vesicularparticles is injected to the region near a tumorous tissue of anexaminee for use as a therapeutic agent, exposure of the examinee to analternating magnetic field or ultrasonic waves, within not less than 0.5min. and not more than 36 hr. after the start of injection, can raisethe temperature of a tumorous tissue in the close vicinity of themagnetic vesicular particles.

The manufacturing method of the preparation containing magneticvesicular particles of the invention comprises mixing a lipid membraneconstituents and supercritical carbon dioxide and adding a dispersion ofmagnetic microparticles with an attached organic compound, followed bydischarge of carbon dioxide, whereby the magnetic microparticles arecovered with a lipid membrane to form magnetic vesicular particle(s).

The magnetic vesicular particles are formed preferably under conditionsof a pH of 7 to 10 and a temperature of 3 to 30° C. in the presence ofan organic compound having at least two groups selected from a hydroxylgroup, carboxyl group, carbamoyl group, amino group, mercapto group,sulfo group, dithio group, thiocarboxy group and dithiocarboxy group.

The diagnostic therapeutic system of this invention is a system forperforming diagnosis of a tumor site of an examinee and therapy thereof,using the above-described preparation containing magnetic vesicularparticles. The diagnostic therapeutic system comprising:

an automatic injection device to automatically inject the preparationcontaining magnetic vesicular particles into an examinee,

a diagnosis device provided with a first exposure section to subject thepreparation-injected examinee to a first exposure to an ultrasonic, anelectromagnetic wave or an X-ray, and an imaging section to scan a tumorsite in which the magnetic vesicular particles have been accumulated bythe first exposure,

a therapy device provided with a second exposure section to subject themagnetic vesicular particle-accumulated tumor site to a second exposureto an alternating magnetic field or an ultrasonic, and a temperaturemeasurement section to measure the temperature of the tumor site andthat of a normal site near the tumor site during the second exposure toan alternating magnetic field or an ultrasonic, and

a control device which is connected to each of the automatic injectiondevice, the diagnosis device and the therapy device through a network,controls the operation of each of these devices and performs controlamong these devices.

The above-described temperature measuring section preferably conductstemperature measurement which is non-invasive for the examinee and thenon-invasive temperature measurement calculates the temperature from avertical relaxation time by a signal intensity method, a proton chemicalshift by a phase method or a diffusion coefficient by a diffusion imagemethod, each of which uses a nuclear magnetic resonance imagingapparatus, or from values obtained in microwave radiometry using pluralfrequencies.

It is preferred that the temperature measurement section measures thetemperature of a tumor site and that of a normal site near the tumorsite and successively transmits measurement results to a control device,and when the control device confirms from the received measurementresults that the tumor site has risen to a prescribed temperature, thecontrol device transmits an instruction to stop the exposure to thealternating magnetic field or the ultrasonic to the second exposuresection, thereby controlling therapy.

It is also preferred that the second is so controlled that the secondexposure section exposes the magnetic vesicular particle-accumulatedtumor site to an alternating magnetic field or an ultrasonic, while thefirst exposure section exposes the tumor site to an ultrasonic, anelectromagnetic wave or an X-ray and the imaging section scans themagnetic vesicular particle-accumulated tumor site to confirm thelocation of the tumor site, whereby therapy is performed with confirmingthe tumor site.

The magnetic vesicular particles contained in the preparation of thisinvention enables selective delivery to a disease site or a tumor focus(targeting capability). Accordingly, the preparation is not onlyapplicable to a therapeutic agent, a contrast medium and a drugtransporting carrier but is also usable in thermotherapy employingheat-generation, and further usable in diagnosis and therapy of cancerby combinations of the foregoing.

According to the manufacturing method of this invention, the preparationcontaining magnetic vesicular particles can be made without using anyorganic solvent so that the obtained preparation exhibits high safety toa living body.

In the diagnostic therapeutic system of this invention, examination,diagnosis and therapy of a disease site or a tumor focus can beconducted as a single system. Accordingly, diagnosis and therapy whichhave conventionally been conducted separately, can be done concurrentlyor continuously, thereby lessening the burden given to the patient.

BRIEF EXPLANATION OF THE DRAWINGS

FIG. 1 illustrates a magnetic vesicular particle contained in thepreparation of the invention, and plural magnetic microparticles areenclosed within the grain.

FIG. 2 also illustrates the magnetic vesicular particle.

FIG. 3 illustrates an embodiment of a diagnostic therapeutic systememploying a preparation containing magnetic vesicular particles.

PREFERRED EMBODIMENTS OF THE INVENTION

The preparation of this invention contains magnetic vesicular particlesand optionally auxiliary agents.

As shown in FIG. 1, magnetic vesicular particle 1 is formed by coveringmagnetic microparticle(s) 2 with lipid membrane 4, i.e., by includingthe magnetic microparticles within the lipid membrane, in which organiccompound 3 having at least two groups selected from a hydroxyl group, acarboxyl group, a carbamoyl group, an amino group, a mercapto group, asulfo group, a dithio group, a thiocarboxyl group and a dithiocarboxylgroup (hereinafter, also denoted simply as organic compound 3) is bondedto the magnetic microparticle(s) 2 though a chemical bond. FIG. 1 showsan embodiment of the magnetic vesicular particle in which pluralmagnetic microparticles are included within a lipid membrane, but asingle magnetic microparticle may be included within a lipid membrane.In one aspect, the magnetic vesicular particles satisfy the followingequation:0.05≦R/(r×100)≦1.5

wherein R represents an average grain size of the magnetic vesicularparticles and r represents an average particle size of the magneticmicroparticles.

In the magnetic vesicular particle 1 used in this invention, as shown inFIG. 2, the magnetic microparticle(s) 2, chemically bonded to theorganic compound 3, are covered with the lipid membrane 4, the surfaceof which is preferably bonded via linkage material 7 to physiologicallyfunctional material 5 such as an antibody or antitumor-active material6. Although FIG. 2 shows an embodiment of the magnetic vesicularparticle in which plural magnetic microparticles are covered with lipidmembrane, a single magnetic microparticle may be covered with lipidmembrane. In FIG. 2, a physiologically functional material and anantitumor-active material are bonded but there may also be bonded atleast one physiologically active material selected from physiologicallyfunctional materials, additionally stabilizing material, medicinallyactive material, medicinally active chelate material, antitumor-activematerial, immunopotentiation material, cell fusion material and genetransfer mediator material to be described later. As a result of bondingto physiologically active material, the magnetic vesicular particles aredelivered to the tumor site not only for detection but also exerts aspecific effect on tumorous tissue. Accordingly, it is usable as acontrast medium exhibiting superior detectability for tumorous tissueand in thermotherapy using energy exposure such as exposure to analternating magnetic field or exposure to a ultrasonic wave, it raisesthe temperature of tumorous tissue in the close vicinity of the magneticvesicular particles and is also usable as a therapeutic agent which iscapable of allowing physiologically active material to act onto thetumorous tissue. FIGS. 1 and 2, each illustrates a magnetic vesicularparticle contained in the preparation of this invention but specificembodiments are by no means limited to these.

In the specification, “cancer” refers to a malignant tumor and it isalso referred to simply as “tumor”. The expression, being enclosedwithin lipid membrane or liposome membrane means being included in theliposome membrane or liposome and associated with the lipid membrane, orexisting in a water phase (internal water phase) enclosed in theinterior of the lipid membrane.

Magnetic microparticles usable in this invention can use, as a maincomponent, any one of magnetite, Fe₂O₃, Fe₃O₄, mixed ferrite, and otheriron-containing compounds including organic ferromagnetic material. Ofthese, ferrite, Fe₃O₄ exhibiting a maximum force, which is superior inmagnetic responsibility, is specifically preferred. There was developeda technique in which nano-sized microparticles of ferrite (solidsolution of Fe₃O₄ and δ-Fe₂O₃) exhibiting superior magneticcharacteristics were synthesized by a controlled precipitation methodunder mild conditions of a temperature of 4 to 25° C. and a neutral pH,as described in JP-A No. 2002-128523. The preparation of this inventionemploys such mixed ferrite microparticles as suitable magneticmicroparticles. Magnetic microparticles having the foregoing ferrite asa core can further contain various metal elements such as Zn, Co and Nito control magnetic characteristics.

The average particle size of the magnetic microparticles is usually from1 to 30 nm, preferably from 5 to 25 nm, and more preferably from 5 to 20nm.

The magnetic vesicular particles, each contains at least one magneticmicroparticle as a ferrite core. The number of magnetic microparticlesis variable depending on the average size of magnetic microparticles,the average size of magnetic vesicular particles and magneticcharacteristics required as the preparation of this invention,therefore, the number of magnetic microparticles is optimally adjusted.

Regarding the average grain size of magnetic vesicular particles havingat least one magnetic microparticle, it is necessary to take sizing ofthe grain size into account to maintain passive targeting capability.Japanese Patent No. 2619037 describes that removal of liposomes ofparticle sizes of 3000 nm or more can avoid embolization of pulmonarycapillaries. However, liposomes of sizes of 150 to 3000 nm are notalways antitumorous. It therefore needs to optimally design the particlesize according to the objective of the contrast medium. In thisinvention, the average grain size of magnetic vesicular particles isusually from 50 to 300 nm, preferably from 50 to 200 nm, and from 50 to150 nm. For example, to achieve selective delivery to the tumor site,the average grain size from 80 to 150 nm is specifically preferred.Making the grain size uniform within 100 to 200 nm (preferably 110 to130 nm) enables to allow magnetic vesicular particles to be selectivelyconcentrated to cancerous tissue (EPR effect). Pores of neovascularwalls existing in solid cancerous tissue are extraordinarily large, ascompared to the pore size of capillary wall fenestra of normal tissue,30 to 80 nm and even molecules having a size of ca. 100 nm to ca. 200 nmleak from the vascular wall. The EPR effect is due to the fact that aneovascular wall existing in cancerous tissue exhibits higherpermeability than a microvascular wall, so that blood retentioncapability have to be enhanced. The magnetic vesicular particles are notso large and are difficult to become a target of capture by areticulated endothelial cell.

In cases when the preparation of this invention is used as a contrastmedium, the average size of magnetic vesicular particles which fallswithin the foregoing range results in enhanced detection capability ofcancerous tissue. On the other hand, in cases when the preparation ofthis invention is used as a therapeutic agent for use in thermotherapyfor tumor and when an average grain size falls within this range, thetemperature of tumorous tissue close to the magnetic vesicular particlesis limitedly raised upon energy exposure, substantially withoutaffecting normal tissue. When the energy exposure is exposure to analternating magnetic field, the average size of magnetic microparticlesis preferably not less than 10 nm in terms of rotatability of themagnetic microparticles under the alternating magnetic field. An averageparticle size of less than 10 nm results in poor rotation of themagnetic microparticles, leading to a deteriorated temperatureincreasing efficiency. Specifically, the average particle size isusually from 10 to 30 nm, preferably from 10 to 25 nm, and morepreferably from 10 to 20 nm.

Magnetic microparticles are desirably as large as possible from theviewpoint of contrast performance (specifically, T2 relaxation time) ofa contrast medium for use in the nuclear magnetic resonance imaging orsimply magnetic resonance imaging (MRI) or from the viewpoint offunction as a heat-generating element of thermotherapy, with consideringthat the particle size and the magnetic moment are dependent on eachother, affecting their effects. On the other hand, the grain size ofmagnetic vesicular particles is limited to be not more than a prescribedsize from various biological characteristics, specifically, targetdirectionality. Accordingly, as a preferable range of the average sizeto find a compromise point between both parameters, the magneticvesicular particles are required to satisfy the following equation,indicating the relationship between the particle size of magneticmicroparticles and the grain size of magnetic vesicular particles:0.05≦R/(r×100)≦1.5,preferably, 0.05≦R/(r×100)≦1.0, andmore preferably, 0.05≦R/(r×100)≦0.8,wherein R represents the average grain size of magnetic vesicularparticles and r represents the average particle size of magneticmicroparticles included in the magnetic vesicular particles.

When the magnetic vesicular particles satisfy the foregoing equation,sizes of constituent magnetic microparticles and the size of the wholeparticles are guaranteed to fall within the suitable range. Thereby, themagnetic vesicular particles can be delivered specifically to the tumorsite. As a result, the magnetic vesicular particles are usable as acontrast medium capable of enhancing detection capability or usable as atherapeutic agent capable of limitedly raising the temperature oftumorous tissue in the close vicinity of the magnetic vesicularparticles in thermotherapy accompanying with energy exposure such asexposure to an alternating magnetic field or exposure to a ultrasonicwave.

The organic compound chemically bonded to magnetic microparticles is onewhich has in its molecule at least two binding groups (a) selected fromhydroxyl group (—OH), carboxyl group (—COOH), carbamoyl group (—CONH₂),amino group (—NH₂), mercapto group (—SH), sulfo group (—SO₃H), dithiogroup (—SS—), thiocarboxyl group [—C(S)OH or —C(O)SH] and dithicarboxylgroup (—CSSH). Herein, the expression, chemically bonded means beingbonded through chemical bond. In general, the chemical bond isclassified to covalent bond, ionic bond, metallic bond and coordinatebond (including chelate bond). The organic compound may be bonded to themagnetic microparticles through any of the foregoing chemical bonds.These groups (a) are each chemically bonded to the magneticmicroparticle surface, whereby polyvalent bonding effects of the organiccompound are displayed. Thus, the organic compound is bonded to themagnetic microparticles through at least two binding groups, formingstrong linkage to the magnetic microparticles. Such a poly-bindingorganic compound allows plural magnetic microparticles to be connectedto each other and is further bound to lipid membrane 4 covering magneticmicroparticle(s) 2, playing the role as a connector. As a result,magnetic vesicular particles are entirely structurally stabilized.Magnetic microparticles can stably be enclosed within lipid membrane bythe existence of organic compound 3. In one case, the organic compoundbonding to the magnetic microparticle may be linked at the other end tophysiologically active material or medicinally effective material.Lipophilic physiologically active material or medicinally effectivematerial which is delivered by magnetic vesicular particles as a carrieris expected to preferably interact with lipid membrane 4 coveringmagnetic microparticle(s) 2.

Such organic compound is not specifically limited if it has at least twobinding groups (a). The binding groups (a) may be an identicalfunctional group or may be a combination of different functional groups.Specific examples thereof include an organic compound which contains ahydrogen atom or an organic group (b) at the prescribed carbon positionof compound (A) having at least two binding groups (a). A compound whichhas a branched structure at least one end of the compound skeleton ispreferred.

Examples of a compound forming a basic skeleton of the compound (A)include phthalic acid, isophthalic acid, terephthalic acid, succinicacid, glutaric acid, adipic acid, pimelic acid, fumaric acid, maleicacid, 2-mercaptoamine, 6-aminehexanethiol, 2-mercaptopropionic acid,asparagic acid, glutamine, malic acid, oxaloacetic acid, 2-ketoglutaricacid, serine, threonine, cysteine, cysteic acid, cystine,N-acetylcysteine, cysteine ethyl ester, and dithiosraytol. A singlecompound selected from the foregoing or a combination of two or morecompounds are usable.

The above-described organic group (b) is not specifically limited andexamples thereof include an alkyl group, an aralkyl group, an alkoxygroup, an aryl group, an alkyleneoxy group, an aliphatic hydrocarbongroup, and a polyoxyalkylene group, each of which may be substituted.The organic compound preferably plays a role in combining magneticmicroparticles and the lipid membrane by allowing the organic group (b)and the lipid membrane to interact with each other via various types ofbonding, such as a covalent bond, ionic bond, hydrophobic bond, andhydrogen bond. From such a point of view, the organic group (b) ispreferably a hydrophobic, medium to a long chain alkyl group ordissociative group which is capable of interacting with the phospholipidof the lipid membrane. It is therefore desirable to form a side-chain ofthe compound (A) using a straight chain aliphatic hydrocarbon grouphaving 3 to 30 carbon atoms or a polyalkyleneoxy group of these organicgroups (b).

An organic compound which has an organic group (b) at the prescribedcarbon atom position of the compound (A) can be prepared byconventionally known methods.

As shown in FIG. 2, organic compound 3 is chemically bonded to magneticmicroparticle 2 via at least two binding groups (a) and lipid membrane 4is formed together with a part of organic group (b), whereby the lipidmembrane is strongly bonded. As the magnetic microparticle(s) are bondedto lipid membrane 4 via an organic compound, stability within a humanbody or during storage is enhanced. Even when subjected to energyexposure in thermotherapy, magnetic vesicular particles are not degradedso that thermotherapy can be efficiently conducted.

Such magnetic microparticles bonded to an organic compound can be madeby forming magnetic microparticles in the presence of an organiccompound under prescribed conditions.

Specifically, an aqueous solution containing metal ions such as Fe²⁺ isdropwise added to an aqueous solution or dispersion of an organiccompound containing at least two functional groups described above andmixed with stirring. It is preferred to control the reaction mixture ata pH of 7 to 10 and a temperature of 3 to 30° C. so as not to impair theactivity of the organic compound. The pH can be adjusted using a buffersolution of ammonium acetate, potassium acetate, ammonium chloride,ammonium hydroxide or their mixture. Oxidation is performed under mildconditions to such an extent that aerial oxygen is brought in withstirring, so that the organic compound can be fixed to the ferritewithout altering the organic compound or impairing its activity.Magnetic microparticles formed under such mild conditions arehomogeneous in composition and uniform in magnetic characteristics.Oxidation may be performed by combining a commonly known method and theuse of nitrous acid or hydrogen peroxide with the foregoing method.

When an aqueous solution containing metal ions is dropwise added, themetal ions are adsorbed to a prescribed group of the organic compoundand a part of Fe²⁺ ions is oxidized to Fe³⁺ ions to cause formation of aspinel ferrite layer. On the surface of the spinel ferrite layer,hydroxyl groups are formed and further thereon, Fe²⁺ ions are adsorbedand oxidized to form a new spinel ferrite layer. This process isrepeated to perform growth of ferrite, whereby magnetic microparticlesattached to the organic compound are formed. Various types of ferritescan be made by addition of other metal ions such as Co²⁺, Ni²⁺ and Zn²⁺,in addition to Fe²⁺. The reaction is undergone until magneticmicroparticles reach a prescribed size. In this invention, magneticmicroparticles can be made by allowing an organic compound havingorganic group (b) bonded to the basic skeleton of compound (A) to existin advance. Alternatively, magnetic microparticles can be formed in thepresence of only an organic compound having the basic skeleton ofcompound (A), followed by bonding organic group (b) to compound (A).

It is also preferred that particulate ferrite as a core of the magneticmicroparticle is dispersed in an aqueous solution or in a dispersion ofan organic compound and according to the foregoing method, a spinelferrite layer bonded to the organic compound is formed on the surface ofthe particulate ferrite. The thus prepared magnetic microparticles whichare chemically bonded to the organic compound, are comprised of a coreof a ferrite having a uniform crystal structure. The use of suchmagnetic microparticles as a contrast medium in MRI examinations canachieve accurate imaging.

The thus prepared magnetic microparticles to which an organic compoundis chemically bonded, are obtained in the form of a dispersion. Whenusing the thus obtained magnetic microparticles in the manufacture ofmagnetic vesicular particles, it is preferred that the magneticmicroparticles are refined by the prescribed method and dispersed in anaqueous medium. As an aqueous medium is employed water such as distilledwater, official water for injection or pure water, physiological saline,various buffer solutions and an aqueous salt-containing solution.

In the constitution of the magnetic vesicular particle, magneticmicroparticle(s) 2 which are bonded to organic compound 3 are coveredwith a lipid membrane via the organic compound (as shown in FIGS. 1 and2). Covering magnetic microparticles with a lipid membrane allows theparticles to be dispersed in an aqueous medium and makes it feasible toform a magnetic vesicular particle-containing preparation exhibitingsuperior dispersion stability. Magnetic microparticles exhibiting arelatively high residual magnetism often cause magnetic coagulation,forming precipitates in medium. The lipid membrane is inherentlybiocompatible and exhibits enhanced affinity to tissue, andspecifically, directionality to hydrophobic tissue is provided tomagnetic microparticles. Physiologically active material, medicinallyeffective material or the like can be advantageously added throughdesignation of lipid membrane constituents, modification of the membranesurface and the like.

Lipid membrane covering magnetic microparticles is generally lipidmulti-layer membrane, preferably a multi-layer membrane formed of anamphiphilic molecule having a medium or long chain aliphatic acidresidue or a medium or long chain alkyl group and a hydrophilic group.Phospholipid and/or glycolipid are preferably used as such a lipidmembrane constituent. A vesicle constituted of lipid bilayer (mainlycomposed of phospholipid) is generally referred to as “liposome”.

Representative examples of a phospholipid include phosphatidylcholine,phosphatidylserine, phosphatidylethanolamine, phosphatidylglycerol,phosphatidylinositol, phosphatidic acid, cardiolipin and sphingomyelin.There are also usable phospholipids derived from plants and animals suchas egg yolk or soybeans and their hydrogenation products or hydroxidederivatives, so-called semi-synthetic phospholipids. Fatty acidsconstituting a phospholipid are not specifically limited, and saturatedand unsaturated fatty acids are usable.

Specific examples of neutral phospholipid includedipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine(DSPC), dimyristoylphosphatidylcholine (DMPC),dioleylphosphatidylcholine (DOPE), dimyristoylphosphatidylethanolamine,dipalmitolphosphatidylethanolamine, anddistearoylphosphatidylethanolamine.

In this invention, the lipid membrane preferably forms a liposome. Thus,the magnetic vesicular particles of this invention preferably areliposomes including one or more magnetic microparticles within lipidmembrane vesicles. The liposomes preferably exhibit a positive surfacecharge; in other words, liposome vesicles preferably have the cationicsurface. To make the liposome surface charge positive, it is preferredto use, together with the foregoing neutral phospholipid, at least oneselected from a cationic phospholipid, a cationic lipid and along chaincationic compound compatible with a phospholipid. Cationic surfacecharge of liposome membrane enables specific introduction of magneticvesicular particles contained in the preparation into a negativelycharged tumor cell.

Examples of cationic phospholipid include an eater of phosphatidic acidand aminoalcohol, such as an ester of dipalmotoylphosphatidic acid(DPPA) or distearoylphosphatidic acid, and hydroxyethylenediamine.Examples of cationic lipids usable in the invention include1,2-dioleoyloxy-3-(trimethylammonium)propane (DOTAP),N,N-dioctadecylamidoglycylspermine (DOGS), dimethyloctadecylammoniumbromide (DDAB), N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammoniumchloride (DOTMA),2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propaneaminiumtrifluoroacetate(DOSPA) andN-[1-(2,3-dimyristyloxy)propyl]-N,N-dimethyl-N-(2-hydroxyethyl)ammoniumbromide (DMRIE). Examples of a long chain cationic compound include atleast 10 carbon atoms containing onium salts such as ammonium salt orphosphonium salt.

Examples of glyceroglycolipids include glycerolipids such asdigalactosyldiglyceride and digalactosyldiglyceride sulfuric acid ester;sphingoglycolipids such as galactosylceramide, galactosylceramidesulfuric acid ester, lactosylceramide, ganglioside G7, ganglioside G6and ganglioside G4.

To combine a physiologically active material with the foregoingphospholipid, a functional group capable of being bonded to aphysiologically active material may be introduced within a range notdeviating from the object of this invention. In addition to theforegoing lipid, other material may optionally be incorporated as aliposome membrane constituent. Examples thereof include glycols such asethylene glycol and propylene glycol, sterols acting as a membranestabilizer such as cholesterol, dihydrocholesterol and cholesterolester. Sterol derivatives are also effective for stabilization ofliposome, as described in JP-A No. 5-245357. Of these, cholesterol isspecifically preferred.

Sterols are used usually in an amount of from 0.05 to 1.5 parts byweight, preferably from 0.2 to 1 parts by weight and more preferablyfrom 0.3 to 0.8 parts by weight per part by weight of phospholipid. Anamount of less than 0.05 parts by weight does not achieve stabilizationby the sterol to enhance dispersibility of mixed lipids, while an amountof more than 2 parts by weight inhibits liposome formation or results inunstable formation thereof.

Other additive compounds include, for example, phosphoric acid dialkylesters as negative-charged material, e.g., diacetyl phosphate, andaliphatic amines as a compound providing a negative charge, such asstearylamine.

In this invention, polyethylene glycol (hereinafter, also denoted simplyas PEG) can be used as one constituent for liposome covering magneticmicroparticles. Thus, attachment of PEG to the liposome can provide arole as “linkage material” as described later or a new function to theliposome. For example, such a PEG-modified liposome can be expected tohave an effect of having a hydrophilic tendency, becoming lessrecognizable from an immune system or increasing blood stability.Specifically, since lipid components easily accumulate in liver, the useof a liposome containing no PEG or trace amounts of PEG is desired. Inthe case of integrating other organs, the liposome becomes a state ofbeing stealthy by introduction of a PEG, becoming difficult to begathered in the liver, therefore, the use of a PEG-modified liposome isrecommended. Introduction of a PEG forms a hydration sphere, therebystabilizing the liposome and enhancing blood retention. Functions can beadjusted by changing a length of oxyethylene units of a PEG and itsintroducing ratio. Polyethylene glycol having 10 to 3500 (preferably,100 to 2000) oxyethylene units is preferred as PEG. A PEG is preferablycontained in an amount of 0.1% to 30% by weight, and more preferably 1%to 15% by weight, based on the lipid constituting the liposome.

A cholesterol enclosed in the liposome membrane is capable offunctioning as an anchor to introduce a polyalkylene oxide.Specifically, cholesterol contained as a liposome membrane constituentin the membrane may optionally be attached to a polyalkylene oxide groupvia a linker. A short chain alkylene or oxyalkylene group is used as alinker. JP-A No. 9-3093 discloses novel cholesterol derivatives, inwhich various functional substances can be efficiently fixed at the topof a polyoxyalkylene chain, which can then be employed as a liposomeconstituent.

PEG-modification of a liposome can be accomplished using commonly knowntechniques. For example, a polyethylene glycol (PEG) group linked to ananchor compound (e.g., cholesterol, phospholipid) is mixed with aphospholipid as a membrane constituent to form a liposome and the anchorcompound may be allowed to be linked to an activated PEG group. Sincethe PEG group introduced onto the liposome surface is unreactive to“physiologically active material” to be described later, it is difficultto fix the physiologically active material onto the liposome surface.Instead thereof, PEG, the top of which has been chemically modified isbonded to a phospholipid, which is included as a liposome constituent toprepare liposomes.

In place of polyethylene glycol (PEG), commonly known polyalkylene oxidegroups may be introduced, which is represented by general formula:-(AO)_(n)—Y where AO is an oxyalkylene group having 2 to 4 carbon atoms,n represents a mean addition molar number and is a positive number of 1to 2000, and Y is a hydrogen atom, an alkyl group or a functional group.Examples of an oxyalkylene group (represented by “AO”) having 2 to 4carbon atoms include an oxyethylene group, oxypropylene group,oxytrimethylene group, oxytetramethylene group, oxy-1-ethylethylenegroup and oxy-1,2-dimethylethylene group.

In the foregoing, n is 1 to 2000, preferably, 10 to 500, and morepreferably 20 to 200. When n is 2 or more, plural oxyalkylene groups maybe the same or differ. In the latter case, differing oxyalkylene groupsmay be in a random form or in a block form. To provide hydrophilicity toa polyalkylene oxide group, ethylene oxide alone, as AO is preferablyaddition-polymerized, in which n is preferably 10 or more. In cases whendifferent alkylene oxides are addition-polymerized, it is desirable thatat least 20 mol % (preferably at least 50 mol %) of ethylene oxide isaddition-polymerized. To provide lipophilicity to an oxyalkylene group,it is preferred to increase the molarity of alkylene oxide(s) other thanethylene oxide. For example, a liposome containing a block copolymer ofpolyethylene oxide and polypropylene oxide (or polyethyleneoxide-block-polypropylene oxide) is a preferred embodiment of thisinvention.

The designation Y is a hydrogen atom, an alkyl group or a functionalgroup. The alkyl group includes an aliphatic hydrocarbon group having 1to 5 carbon atoms, which may be branched. The functional group of theforegoing Y is to attach functional material such as sugar,glycoprotein, antibody, lectin and a cell adhesion factor to the top ofa polyalkylene oxide group and examples thereof include an amino group,oxycarbonylimidazole group and N-hydroxysuccinimide.

The polyalkylene oxide group plays the role of linkage material, asdescribed later, similarly to polyethylene glycol. The liposomeanchoring a polyalkylene oxide chain, to the top of which the foregoingfunctional material is bonded, not only exhibits effects due tointroduction of a polyalkylene oxide group but also gives full play offunctions of the functional material, for example, a function as arecognition element, such as directivity to a specific organ andcancerous tissue directivity.

A phospholipid or cholesterol which contains a polyalkylene oxide groupcan be used alone or in combinations thereof. The content thereofpreferably is 0.001 to 50 mol %, more preferably 0.01 to 25 mol %, andstill more preferably 0.1 to 10 mol %, based on the total amount ofliposome membrane forming components. A content of less than 0.001 mol %results in reduced expected effects.

Introduction of a polyalkylene oxide chain into liposomes can employcommonly known techniques. Thus, an anchor bonding a polyalkylene oxide(e.g., cholesterol, phospholipid) is mixed with phospholipid as amembrane constituent to form liposomes and an activated polyalkyleneoxide may be attached to the anchor. This method needs to performmultistage chemical reaction on the liposome membrane surface aftercompleting formation of liposomes. As a result, an introducing amount ofan objective physiologically active material is limited to the lowerside and contamination with reaction by-products or impurities iscaused, causing further problems such as marked damage of the liposomemembrane.

As a preferred manufacturing method replacing this, phospholipidpolyalkylene oxide derivative is included in advance in phospholipids asraw material to form liposomes.

For examples, JP-A No. 7-165770 proposed polyethylene oxide (PEO)derivatives of a phosphatidylamine and the like, such asdistearoylphosphatidyldiethanolamine polyethyleneoxide (DSPE-PEO).Further, JP-A No. 2002-37883 discloses an extremely purified,polyalkylene oxide-modified phospholipid to prepare a water-solublepolymer-modified liposome exhibiting enhanced blood retentivity. It isalso disclosed that the use of a polyalkylene oxide-modifiedphospholipid having a relatively low monoacyl content in the preparationof liposome leads to superior aging stability of liposome dispersions.

Linkage material as a linker allows various physiologically activematerials or medicinally effective materials to be bonded in theinterior of the magnetic vesicular particle. When a physiologicallyactive material or medicinally effective material having a relativelylow molecular weight, as a ligand, is linked to an acceptor, itsapproach to the ligand is often hindered by steric hindrance due to themagnetic vesicular particle. Such hindrance can be avoided by allowing alinkage material as a spacer having an appropriate length to intervenebetween the magnetic vesicular particle and a ligand. Preferred examplesof a linkage material include a polyethylene glycol chain and apolyalkylene oxide chain, such as ethylene glycol diglycidyl ether(EGDE). A suitable hydrocarbon chain is one which may contain aheteroatom (e.g., oxygen, nitrogen, sulfur, phosphorus). Such ahydrocarbon chain preferably has 2 to 50 carbon atoms (more preferably 3to 40 carbon atoms, and still more preferably 4 to 30 carbon atoms),which may be substituted by a functional group, an alkyl group or anaryl group. There may be further provided a binding group such as a thiogroup, epoxy group, amino group, carboxyl group, histidinetagavidine,streptavidin, and biotin. There may be provided oligonucleotide (no. ofnucleic acid base: 3-100) or polypeptide (no. of nucleic acid base:3-50) which is modified with an amino group, carboxyl group or thiolgroup.

The linkage material may be one member of lipid membrane constituents,as a lipid derivative constituting the lipid membrane.

The magnetic vesicular particles are each bonded to physiologicallyactive material via the linkage material (7) or the organic compound 3,as described above. In the case of liposome-magnetic vesicularparticles, physiologically active material may be directly bonded ontothe lipid membrane surface of the liposome. Alternatively,physiologically active material is bonded to the organic compound 3 andexists in the interior of the lipid membrane. Examples ofphysiologically active material include physiologically functionalmaterial, additively stabilizing material, medicinally active material,medicinally active chelating material, antitumor-active material,immunopotentiating material, cell fusion material, and gene transfermediating material. It is also feasible that the foregoingphysiologically active material is allowed to bond to magnetic vesicularparticles and concentrate selectively to the targeted site through amagnetic operation.

Examples of physiologically functional material include physiologicalmaterial, such as sugar, glycoprotein, aptamer, antibody, antigen,lectin, cytokine, growth factor, adjustment factor, physiologicallyactive peptide, cell adhesion factor or hormone; and material displayingphysiological function, such as metabolic material or an alkaloid.

The antibody may be either a polyclonal antibody or a monoclonalantibody. There are exemplified antibodies attached to various kinds ofglycoproteins. Examples thereof include CD44, CD54, CD56 and Fas.Further, an antigen which is expressed specifically in cancer cells andan antibody against tumor-related antigens are also desirable. In thisregard, antigens are commonly known, such as MN, HER2, MAGE3, VEGF andCEA.

An antibody against “WTI protein”, which does not exist in a normal cellbut exists mainly in various kinds of cancer cells or a blood cancercell, is also cited as a preferred antibody. It is proved that a part ofthe WTI protein (nine aminoacid-WTI peptides) links to HLA moleculeexisting on the surface of cancer cell, which becomes a marker of thecancer cell. The foregoing antibody can be prepared by the conventionalmethod, using WTI peptide.

The additively stabilizing material is a material capable of stabilizinga structure of magnetic microparticles which are associated bysolvention. Examples thereof include polyvinyl alcohol, polyvinylpyrrolidone, polyethylene glycol, polyalkylene oxide, dextran, cellulosederivatives, muco-plysaccharide, protein, polypeptide, polyaminoacid,and polynucleotide.

The medicinally active material is a compound exhibiting bioactivity ormedicinal effectiveness. Examples thereof include antitumor-activematerial, anti-infective material, antiviral, antibiotic,anti-inflammatory compound, chemotherapeutic agent, circulatorymedicine, alimentary drugs, and neural medicine.

The medicinally active chelating material is material detoxificationupon chelate formation or stabilizing action upon chelation. Examplesthereof include EDTA, DTPA, cyclin, polycarboxylic acid, polyamino acid,porphyrin, and catecholamine.

The antitumor-active material is material exhibiting tumor-shrinkageeffects. Examples thereof include antibiotics, plant alkaloids,alkylation agents, antitumor agents contained in antimetabolite,anti-vascularization medicine and tumor neocrotizing factors. Examplesof an anti-vascularization medicine include TNP-470 (AGM-1470,synthesized analog of fungus discharge, named fumagilin, produced byTakeda Chemical Industries Ltd.), and iostatin (Herbert Medical School,Children's Hospital, Surgical Research Lab.), and integlin α_(V)β₃antagonistic drugs (e.g., monoclonal antibody of integlin α_(V)β₃, TheScripps Research Institute, LaJolla, Calif.).

The immunopotentiation material is one exhibiting action to enhanceactivity of immunocytes including lymphocyte and macrophage. Examplesthereof include interferon, Krestin, Picibanil, Lentinan, IFA andOK-432.

Cell fusion material is used in a cell fusion operation and promotescell fusion. Examples thereof include polyalkylene glycol, arylpolyalkylalkylene glycol, arylpolyalkylene glycol, alkylarylpolyalkyleneglycol and their derivatives.

The gene transfer mediating material is one functioning as a carrier forgene transfer and examples thereof include polyalkylene glycol (e.g.,polyethylene glycol), polyimine (e.g., spermine, spermidine,pentaethylenehexaamine, polyethyleneimine, protamine sulfate), virusvectors and plasmid vectors.

The combination of a photosensitizer of liposomes including phospholipidexhibiting a transition temperature, used for photodynamic therapy andcancer thermotherapy is feasible and enhanced therapeutic effects areexpected. A magnetic vesicular particle preparation employing a liposomewhich links or encloses a photosensitizing material such as porphyrins,5-aminolevulinic acid, chlorines and phthalocyanine, is given to apatient, and after an elapse of time and when heated from the outside ofbody, local thermotherapy is conducted using microwaves orelectromagnetic waves.

The preparation containing magnetic vesicular particles, according tothis invention, may optionally contain, as auxiliary agents, apharmaceutically allowable buffering agent, stabilizer, antioxidant suchas α-tocopherol, viscosity-adjusting agents or chelating agents. Theseare optimally employed prevent oxidation reduction reaction oralteration such as coagulation or precipitation. One feature of thepreparation of this invention is that any organic solvent is notsubstantially contained in the included lipid membrane or in theinternal water phase.

Manufacturing methods of the preparation containing magnetic vesicularparticles of this invention are not specifically limited if the magneticmicroparticle surface bonded to the afore-mentioned organic compound canbe covered with lipid membrane. The preparation can also be manufacturedemploying a conventional shaking method. However, in conventionalmethods, the phospholipid needs to be dissolved in chlorinated solventsand unremoved chlorinated solvents are feared to remain in thepreparation, often causing problems with respect to safety for the humanbody. On the contrary, the preparation containing magnetic vesicularparticles of this invention can be manufactured substantially withoutusing organic solvents such as chlorinated solvents. Thus, thepreparation can be made by the method using supercritical carbondioxide. The expression, substantially without using organic solventsmeans that the upper limit of the residual organic solvent concentrationof the preparation is 10 μg/L. In the following, supercritical carbondioxide includes subcritical carbon dioxide.

A preferred method of manufacturing the preparation containing magneticvesicular particles comprises:

the first step in which the lipid membrane constituent described aboveand liquefied carbon dioxide are mixed within a pressure vessel, afterwhich applying heat and pressure to the interior of the vessel formssupercritical carbon dioxide, and while mixing the lipid membraneconstituents and the supercritical carbon dioxide, a dispersion ofmagnetic microparticles chemically bonded to an organic compound isfurther added thereto and mixed, and

the second step in which after obtaining the mixture in the foregoingstep, the interior of the pressure vessel is evacuated to dischargecarbon dioxide, thereby covering (or enclosing) the magneticmicroparticles with lipid membrane to form magnetic vesicular particles,resulting in an aqueous dispersion of magnetic vesicular particles.

The foregoing method preferably further comprises the third step inwhich the aqueous dispersion of magnetic vesicular particles issubjected to filtration using a filter membrane of a pore size of 100 to1000 nm.

The respective steps will be further described as below.

In the first stage, while mixing lipid membrane constituents andliquefied carbon dioxide in a pressure vessel, the pressure and thetemperature in the interior of the vessel are adjusted so that thecarbon dioxide becomes supercritical, and the lipid membraneconstituents and the supercritical carbon dioxide are mixed.

In the manufacturing method of this invention, the pressure suitable forcarbon dioxide in a supercritical state is from 50 to 500 kg/cm² butpreferably from 100 to 400 kg/cm². The temperature suitable for thesupercritical carbon dioxide is from 25 to 200° C., preferably from 31to 100° C., and more preferably from 35 to 80° C. It is preferred tomaintain the supercritical state by the combination of temperature andpressure within the foregoing range. Stirring conditions are notspecifically limited and suitable ones are appropriately chosen.

Subsequently, while mixing lipid membrane constituents and supercriticalcarbon dioxide, a dispersion of magnetic microparticles which arechemically bonded to an organic compound, is further added thereto andmixed. The dispersion may be added all at once or intermittently withmaintaining the supercritical state.

In the second stage, after obtaining the mixture in the foregoing step,the interior of the pressure vessel is evacuated and carbon dioxide isdischarged, whereby an aqueous dispersion of magnetic vesicularparticles (or vesicles) in which the magnetic microparticles are coveredwith lipid membrane.

The organic compound bonded to the magnetic microparticles has theafore-mentioned organic group (b), a part of which is enclosed in theformation of lipid membrane and connected to the lipid membrane. Thus,the magnetic microparticles and the lipid membrane are tightly bondedvia the organic compound. Accordingly, stability within the human bodyor during storage is enhanced and for instance, the magnetic vesicularparticles are not degraded even when subjected to energy exposure inthermotherapy, performing efficient thermotherapy. In this stage, one orplural magnetic microparticles are enclosed (or covered) with lipidmembrane, whereby an aqueous dispersion of magnetic vesicular particlesis obtained.

In the third stage, the aqueous dispersion of magnetic vesicularparticles, obtained in the second stage, is filtered using a filtermembrane having a pore size of 100 to 1000 nm. Specifically, the aqueousdispersion is passed through an extruder installed with a filter havinga pore of 100 to 1000 nm, whereby 100-150 nm magnetic vesicularparticles can be efficiently made. Thus, adjustment of size andsize-distribution of the magnetic vesicular particles can achieve theobjective effects of the preparation containing magnetic vesicularparticles.

The magnetic vesicular particles of this invention have such anadvantage that the dose of magnetic microparticles, in other words, thedosage of lipid can be reduced, compared to magnetic vesicular particlescomprised of multilamellar vesicles (also denoted simply as MLV). In theconventional manufacturing method of liposomes, MLV of various sizes andforms often exist in a considerable quantity. Accordingly, an operationsuch as ultrasonic exposure or passing through a filter having aprescribed pore size a few times was needed to enhance the proportion ofunilamellar or several lamellar liposomes. On the contrary, in themethod of this invention in which liposomes are formed usingsupercritical carbon dioxide, liposomes of unilamellar orseveral-lamellar vesicles can efficiently be produced, whereby anenhanced rate of enclosing a drug in the liposomes can be achieved.

The thus obtained aqueous dispersion of magnetic vesicular particles isfiltered using the filtration membrane described above to make apreparation containing magnetic vesicular particles, which may befurther subjected to centrifugal separation or ultrafiltration toseparate an aqueous medium from the dispersion, whereby concentrateddispersion is obtained.

The preparation containing magnetic vesicular particles of thisinvention are usable as a medical preparation, i.e., as an imaging agentfor used in examination or diagnosis and also as a therapeutic agent.Specifically, it can be used as a contrast medium such as a radiographiccontrast medium, a contrast medium for use in MRI (nuclear magneticresonance imaging method) or an ultrasonic contrast medium, and as atherapeutic agent for cancer or the like, preferably, a therapeuticagent for use in thermotherapy.

The radiographic contrast medium is given to a lumen region such as avascular tract, a ureter or a uterine tube to be used for examination ofa form or stenosis of the lumen. However, conventionally used compoundsare promptly discharged from the lumen region without interacting withtissue or disease regions, which is not useful for detailed examinationof the tissue or diseased region, specifically such as cancerous tissue.Therefore, an X-ray contrast medium has been desired which can beselectively accumulated in/or onto the targeted tissue or diseasedregion, thereby giving an image which can be distinguished with clearcontrast from the circumference or other regions.

Examination and diagnosis in MRI produce no problem due to radiationexposure and any sectional image of organism can be obtainednon-invasively, which is a rapidly spreading image diagnosis technology.To obtain clear images by enhancing contrast, a contrast medium capableof varying the proton relaxation time is employed in MRI. As a contrastmedium for use in MRI, gadolinium-diethylenetriaminepentaacetic acid(hereinafter, also denoted simply as Gd-DTPA) is only onecontrast-enhancing agent at the present time. The Gd-DTPA contrastmedium which has been introduced into a human body is transferred totissue by the circulating bloodstream. However, the contrast mediumitself has no capability of discriminating tissue.

Ultrasonic image diagnostics usually allow ultrasonic having a frequencyof 1 to 10 MHz to permeate into the interior of the body of an examineevia a converter, on the basis of ultrasonic waves interacting with theinterface of body tissue or body liquid (or humor). An image formed byultrasonic signals is derived from differential reflection/absorption ofultrasonic in the interface. An ultrasonic contrast medium ofmicrocapsules enclosing gas or bubbles is used in this diagnosis method.However, the amount of enclosed gas or bubbles is not always so large,depending on other factors. Accordingly, a sufficient contrast effect isachieved only by a large amount of dose.

The magnetic vesicular particles which have been introduced into thebody as a contrast medium for use in radiography or MRI or as aultrasonic contrast medium can be selectively accumulated in/or onto thetargeted tissue or diseased region, resulting in an image which can bedistinguished with clear contrast from the circumference or otherregions. Thus, the magnetic vesicular particles of this invention can beselectively accumulated in/or onto the targeted tissue or disease regionthrough their grain sizes or surface charge and can provide thecapability of discriminating tissue via physiologically active materialattached to the lipid membrane surface. Furthermore, imaging of tumoroustissue can be effectively realized, due to the fact that the ultrasonicpropagation in magnetic microparticles is faster than that in a livingbody (water). Thus, employing such characteristics, the preparationcontaining magnetic vesicular particles can be used suitably as acontrast medium for use in radiography or MRI, or a ultrasonic contrastmedium.

Specifically, within not less than 1 min. and not more than 48 hr.,preferably not less than 30 min. and not more than 36 hr. after thepreparation containing magnetic vesicular particles of this invention isgiven into a vein of an examinee, a scan is conducted in a ultrasonicimaging diagnosis apparatus, a nuclear magnetic resonance imagingdiagnosis apparatus or an X-ray imaging diagnosis apparatus, therebyachieving enhanced detection of tumorous tissue. The preparationcontaining magnetic vesicular particles may be directly injected neartumorous tissue of an examinee; within not less than 0.5 min. and notmore than 36 hr., preferably not less than 10 min. and not more than 24hr. after the preparation containing magnetic vesicular particles ofthis invention is dosed into a vein of an examinee, scan is conducted ina ultrasonic imaging diagnosis apparatus, a nuclear magnetic resonanceimaging diagnosis apparatus or an X-ray imaging diagnosis apparatus,thereby achieving enhanced detection of tumorous tissue.

The magnetic vesicular particle-containing preparation of this inventionis usable as an imaging agent for examination and diagnosis. Since thepreparation bonds or encloses a physiologically active material,medicinally effective material or an antitumor agent inside or outsidethe lipid membrane, the preparation is also usable as a therapeuticagent for various diseases. The magnetic vesicular particles areaccumulated selectively onto focus such as a diseased region or tumoroustissue. Action of a physiologically active material or medicinallyeffective material is effectively displayed, thereby reducing adverseeffects.

Specific application is exemplified below, but application of thepreparation of this invention is not limited to this.

Thermotherapy for cancer as well as laser therapy or photodynamictherapy belongs to a category of a non-invasive treatment for cancer.Such cancer thermotherapy, which is a treatment having specificcharacteristics and superior faces, has not necessarily been adopted intreatment sites for cancer. Thermotherapy has not been employed alonebut is employed in combination with radiation or an anticancer drug forenhancing effects of the radiation or anticancer drugs. There may becited various reasons therefor. Such therapy has not always achievedsuperior results to other types of treatment. There may be no reason forthis therapy to be replaced by other treatments. There is still room forimprovement of members, drugs and devices.

The present target of this therapy is locally progressive cancer andrecurring cancer which are difficult to be cured in usual treatments.Thermotherapy include warming the whole body (general thermotherapy) andwarming the cancer site or its vicinity (local thermotherapy). Ingeneral, local thermotherapy is mainly conducted using a deviceemploying microwaves, electromagnetic waves (alternating magneticfields) or ultrasonic waves to perform localized warming. A system ofwarming from the outside of the body is most frequently conducted. Thereis also attempted the method of inserting an instrument into a lumensuch as esophagus, rectum, uterus or bile duct and a method insertingseveral electrode needles into cancerous tissue to perform warming.Effects on cancer are achieved at 41° C. or more, and preferably 42.5°C. or more. Cancer near the body surface can readily be warmed to anintended temperature. Cancer localized deep in the body is oftendifficult to be sufficiently warmed due to hindrance of fat, air orbone. Introducing the magnetic vesicular particle-containing preparationof this invention into a body to be used as a heating element forthermotherapy can achieve effective therapeutic effects on canceroustissue in the interior of the body, scattered cancer cells, or minuteinitial cancer cells.

Specifically, when an examinee is subjected to energy exposure withinnot less than 1 min. and not more than 48 hr. (preferably, not less than30 min. and not more than 36 hr.) after the start of dosing of themagnetic vesicular particle-containing preparation of this inventioninto the vein of the examinee, the temperature of cancerous tissue nearthe magnetic vesicular particles is raised and a therapeutic treatmentis performed without adverse effect to normal cells. The preparation mayalso be directly injected near tumorous tissue of the examinee. Exposureof an examinee to energy within not less than 0.5 min. and not more than36 hr. (preferably, not less than 10 min. and not more than 24 hr.)after the start of injection raises the temperature of tumorous tissuenear magnetic vesicular particles. Preferred examples of energy exposureinclude exposure to an alternating magnetic field and exposure toultrasonic waves. The exposure to an alternating magnetic field isconducted preferably at a frequency of 25 to 500 kHz.

Preferred embodiments of a diagnostic therapeutic system using themagnetic vesicular particle-containing preparation of this inventionwill be now be described with reference to a drawing. The embodimentshown in the drawing is one of the embodiments of this invention, butthe invention is by no means limited to this.

As shown in FIG. 3, diagnostic therapeutic system 10 relating to thisinvention comprises:

automatic injection device 20 for automatically giving a preparationcontaining magnetic vesicular particles of this invention to anexaminee,

diagnostic device 30 provided with first exposure section 32 forsubjecting the preparation-given examinee to exposure to an ultrasonicwave, an electromagnetic wave or an X-ray, and imaging section 34 forscanning a tumor site in which the magnetic vesicular particles areaccumulated under the exposure to an ultrasonic wave, an electromagneticwave or an X-ray,

therapeutic device 40 provided with second exposure section 42 ofsubjecting the tumor site in which the magnetic vesicular particles areaccumulated to exposure to an alternating magnetic field or anultrasonic wave, and temperature measuring section 44 for measuring atemperature of the tumor site and that of a normal site near the tumorsite under the exposure to an alternating magnetic field or anultrasonic, and

control device 50 which is connected to the automatic injection device20, the diagnostic apparatus 30 and the therapeutic apparatus 40 throughnetwork 60 and which controls an operation of each of these apparatusesand conducts control among these apparatuses.

As the automatic injection device 20 are usable conventionally knownones. A radiographic imaging diagnostic device, a nuclear magneticresonance imaging diagnostic device or an ultrasonic imaging diagnosticdevice is usable as the diagnostic device 30. A focused ultrasonicheating device or an alternating magnetic field heating device is usableas the therapeutic device 40. Preferably, the automatic injection device20, the diagnostic device 30 and the therapeutic device 40 areintegrated so as to concurrently perform diagnosis and therapy.

Any apparatus which is provided with a control section (CPU), anoperating section such as a keyboard and a mouse and a display sectionsuch as a memory and a display, is applicable as the foregoing controldevice 50, and examples thereof includes a computer. The network 60 maybe connected through information communication network, such as aninternet, LAN (Local Area Network) or WAN (Wide Area Network).Connection of terminals of these is unconcerned with being with wire orwireless.

The diagnostic therapeutic system 10 preferably has a patientinformation database. The control device 50 can access the databaseaccording to its need to obtain information of an examinee, whereby anapproximate position of the tumor site and constitution or healthconditions of the examinee can be confirmed in advance.

In the diagnostic therapeutic system 10, first, the automatic injectiondevice 20 automatically gives a preparation containing magneticvesicular particles to a patient whose tumor site is to be examined andtreated. Specifically, when a patient is taken in the diagnostictherapeutic system, first of all, information for specifying the patientis obtained from an IC tag or a biometric certification such as fingerprints or an iris of the patient. The control device 50 accesses thedatabase based on such information to specify the patient. Then, thecontrol device determines the kind or dose of the preparation containingmagnetic vesicular particles from the data (chart) of the patient andtransmits the automatic injection device 20.

The automatic injection device 20 receives the data such as kind or dosefrom the control device 50 and then doses a preparation containingmagnetic vesicular particles to the patient according to predetermineditems. When completing the dose of the preparation, the automaticinjection device 20 transmits a signal of completion of the dose to thecontrol device 50. Subsequently, the control device 50 transmits asignal of starting diagnosis of the tumor site to the diagnostic device30.

The diagnostic device 30 having received the signal allows the firstexposure section 32 to expose the patient to ultrasonic, electromagneticwave or X-ray and further allows the imaging section 34 to scan thetumor site in which magnetic vesicular particles are accumulated. Theimage data obtained by scanning in the imaging section 34 is transmittedthe control device 50. The control device 50 confirms contrast of theimage based on the previously inputted data to specify the tumor site inwhich magnetic vesicular particles are accumulated. Preferably, thesystem 10 is provided with a display section such as a monitor toartificially confirm the image scanned in the imaging section 34.

The control device 50, which has specified the tumor site, allows thedata regarding the position of the tumor site to be contained in thedatabase of the patient and further transmits the information regardingthe position of the tumor site to the therapeutic device 40. Thetherapeutic device 40 which has received the data regarding the positionof the tumor site, allows the second exposure section 42 to expose thepatient to an alternating magnetic field or an ultrasonic and furtherallows the temperature measuring section 44 to measure the temperatureof the tumor site and the vicinity thereof. In the temperaturemeasurement of the temperature measuring section 44, the temperature invivo is non-invasively measured without embedding a sensor, applyingtemperature variation being represented as variation of resonancefrequency. Preferably, the temperature measuring section 44 conductstemperature measurement which calculates the temperature from a verticalrelaxation time by a signal intensity method, a proton chemical shift bya phase method or a diffusion coefficient by a diffusion image method,each of which uses a nuclear magnetic resonance imaging apparatus, orfrom values obtained in microwave radiometry using plural frequencies.

When the control device 50 judges that the tumor site is too small toperform an effective treatment or judges that a treatment is to beconducted with confirming the tumor site, the control device 50 controlsthe first exposure section 32 to allow the tumor site to be exposed toan ultrasonic wave, electromagnetic wave or X-ray and the secondexposure section 42 to allow the tumor site to be exposed to analternating magnetic field or ultrasonic to perform therapy, whilescanning, by the imaging section, the tumor site in which magneticvesicular particles are accumulated.

The temperature measuring section 44, which has measured the temperatureof the tumor site and a normal site near the tumor site, successivelysends the results thereof to the control device 50. The control device50, which has received the measurement results and confirmed that thetumor site has been raised to a prescribed temperature (e.g., 42° C.),sends the second exposure section 42 an order of stopping the exposureto an alternating magnetic field or an ultrasonic wave. After reachingthe prescribed temperature, the control device may control the secondexposure section 42 so as to continue exposure to an alternatingmagnetic field or an ultrasonic for a given period of time. In caseswhen the tumor site has not reached a prescribed temperature and thenormal site near the tumor site has reached the prescribed temperature,the control device 50 sends the second exposure section 42 an order ofstopping the exposure to an alternating magnetic field or an ultrasonic.

As described above, the diagnostic therapeutic system can performconfirmation of a tumor site to the therapy thereof in a single system.Thus, diagnosis and therapy, which were conventionally conductedseparately, are concurrently performed and lightens the burden on apatient, performing diagnosis and therapy effectively and efficiently.

There has been described the diagnostic therapeutic system of thisinvention but the invention is not limited thereto, and various changesand modifications can be made therein without departing from the objectof this invention.

EXAMPLES

The present invention will be further described with reference tospecific examples but the invention is by no means limited to these.

Organic Compound A

Organic compounds A which is to be added in the course of manufacturingmagnetic microparticles, is prepared in the manner described below.

A hydroxyl group of malic acid and that of tetramethyleneglycolmonomethyl ether were reacted through hexamethylene diisocyanate to formtetramethyleneglycol monomethyl ether-modified malic acid (A-1).Similarly to the foregoing, hexamethyleneglycol monomethylether-modified malic acid (A-2) and decaethyleneglycol monomethylether-modified malic acid (A-3) were prepared. An amino group ofasparagic acid and that of tetramethyleneglycol monomethyl ether werereacted through hexamethylene diisocyanate to form tetramethyleneglycolmonomethyl ether-modified asparagic acid (A-4).

A hydroxyl group of malic acid was reacted with octanoic acid chlorideto form octanoic acid-modified malic acid (A-5). An amino group ofasparagic acid was reacted with stearic acid chloride to form stearicacid-modified asparagic acid (A-6).

The thus prepared organic compound A is as follows:

-   -   A-1: tetramethyleneglycol monomethyl ether-modified malic acid,    -   A-2: hexamethyleneglycol monomethyl ether-modified malic acid,    -   A-3: decaethyleneglycol monomethyl ether-modified malic acid,    -   A-4: tetramethyleneglycol monomethyl ether-modified asparagic        acid,    -   A-5: octanoic acid-modified malic acid,    -   A-6: stearic acid-modified asparagic acid.

Formation of Magnetic Microparticles

Formation Method 1

A 0.1 mol/L ferrous chloride solution and a 0.1 mol/L ferric chloridesolution were mixed with each other at equal volumes to form solution(1). A 28 wt % aqueous ammonia was diluted to 0.01 wt % with distilledwater to obtain solution (2). An aqueous solution containing organiccompound A at a concentration of 1 mmol/L, as shown in Table 1 wasprepared. This solution was adjusted to a pH of 8.4-10.0, using a 1mol/L buffer solution composed of ammonium hydroxide and ammoniumchloride to obtain solution (3).

10.00 ml of the solution (3) was stirred while maintaining a temperatureat 5° C. and blowing air. The solution (1) and solution (2), each of 5.0ml were dropwise added into the solution (3). The addition rate wasadjusted so that the pH was maintained within a range from 7.0 to 8.5with confirming the pH with a pH-meter and the temperature wasmaintained at 5 to 15° C. with monitoring the temperature by atemperature controller. After completion of addition, stirring wascontinued for 1 hr., then, magnetic microparticles were separatedthrough magnetic separation and washed well with distilled water toobtain magnetic microparticles which were chemically bonded to organiccompound A having at least two specific bonding groups. The stirringspeed and the addition rate of the respective solutions (1) and (2) werecontrolled so that the pH and the temperature were maintained with theforegoing range. Magnetic ferrite particles having an average particlesize (r), as shown in Table 1, were thus formed. Using a transmissionelectron microscope, magnetic microparticles were observed to determinetheir particle sizes and the average value of 20 particles was definedas the particle size (r).

Formation Method 2

Similarly to the foregoing formation method 1, solutions (1) and (2)were prepared. An aqueous solution containing an organic compound A at aconcentration of 1 mmol/L, as shown in Table 1 was prepared. Thissolution was adjusted to a pH of 8.4-10.0, using a 1 mol/L buffersolution composed of ammonium hydroxide and ammonium chloride to obtainsolution (3). The aqueous solution described above was adjusted to a pHof 8.4-10.0 to obtain solution (4).

5.00 ml of the solution (4) was stirred while maintaining a temperatureof 5° C. and blowing air thereinto. The solution (1) and solution (2),at 2.5 ml of each, were dropwise added to the solution (4) to preparesolution (5). Subsequently, 5.0 ml of the solution (3) was added to thesolution (5) and further thereto, solutions (1) and (2), 2.5 ml of eachwere added dropwise. The addition rate was adjusted so that a pH of 7.0to 8.5 was maintained with confirming the pH using a pH-meter and thetemperature was maintained at 5 to 15° C. with monitoring thetemperature by a temperature controller. After completion of addition,stirring was continued for 1 hr., then, magnetic microparticles wereseparated through magnetic separation and well washed with distilledwater to obtain magnetic microparticles which were chemically bonded toan organic compound A having at least two specific groups. The stirringspeed and the addition rate of the respective solutions (1) and (2) werecontrolled so that the pH and the temperature of the solution (4) or (5)were maintained with the foregoing range. Magnetic ferrite particleshaving a particle size, as shown in Table 1, were thus formed. Using atransmission electron microscope, magnetic microparticles at the timewhen the solution (5) was prepared were observed to determine theirparticle sizes and an average value of 20 particles was defined asparticle size (r1). Similarly, finally formed magnetic microparticleswere observed and the average value of 20 particles was defined asparticle size (r).

Formation Method 3

Similarly to the foregoing formation method 1, solutions (1) and (2)were prepared. An aqueous solution containing organic compound B at aconcentration of 1 mmol/L, as shown in Table 1 was prepared. Thissolution was adjusted to a pH of 8.4-10.0, using a 1 mol/L buffersolution composed of ammonium hydroxide and ammonium chloride to obtainsolution (3). Subsequently, similarly to the foregoing method 1,solutions were mixed to obtain magnetic microparticles which werechemically bonded to organic compound B having at least two specificgroups.

The magnetic microparticles were dried, placed in hexane and dispersedusing an ultrasonic dispersing machine. To the hexane dispersion of themagnetic microparticles, acid chloride organic compound C) shown inTable 1, which was diluted with hexane to 1 wt %, was added to allow theorganic compound B which was chemically bonded to the magneticmicroparticles and the compound C to react with each other.Subsequently, isopropyl alcohol was added thereto to esterify excessorganic compound C, thereafter, magnetic microparticles were separatedthrough magnetic separation and well washed with acetone and distilledwater to obtain magnetic microparticles which were chemically bonded toorganic compound B and the organic compound was further bonded toorganic compound C. The stirring speed and the adding rate of therespective solutions (1) and (2) were controlled so that the pH and thetemperature were maintained within the foregoing range. Magnetic ferriteparticles having a particle size (r), as shown in Table 1, were thusformed. The particle size (r) was determined similarly to the method 1.

Formation Method 4

Solutions (1), (2) and (4) were each prepared similarly to the formationmethod 2, and solution (3) was prepared similarly to the formationmethod 3, except for the use of organic compound B. Subsequently,magnetic microparticles which were chemically bonded to organic compoundB having at least two specific groups, were prepared similarly to themethod 2.

After the magnetic microparticles were dried, organic compound B, whichwas chemically bonded to magnetic microparticles and compound C werereacted in hexane similarly to the formation method 3. Subsequently,isopropyl alcohol was added thereto to esterify excess organic compoundC, thereafter, magnetic microparticles were separated through magneticseparation and well washed with acetone and distilled water to obtainmagnetic microparticles which were chemically bonded to organic compoundB and the organic compound is further bonded to organic compound C.

The stirring speed and the addition rate of the respective solutions (1)and (2) were controlled so that the pH and the temperature of thesolutions (4) and (5) were maintained within the foregoing range.Magnetic ferrite particles having particle sizes, as shown in Table 1,were thus formed. Particle sizes (r1) and (r) were each determinedsimilarly to the formation method 2.

Magnetic microparticles for comparison in which an organic compound wasnot added in the course of magnetic microparticle formation, wereprepared similarly to the methods 1 to 4.

Formation Method 5

Comparative magnetic microparticles onto the surface of which stearicacid was adhered, were prepared in the following manner. Thus, 1.67 g offerrous sulfate heptahydrate [Fe(II) SO₄.7H₂O] was placed into a samplebottle and dissolved in 8 ml of nitrogen-substituted water. Then, 1 mlof an aqueous sodium nitrite solution (having a concentration of 0.07g/ml) was added, 5 ml of 28 wt % aqueous ammonia was further added andstirred under an atmosphere of nitrogen, while maintaining a temperatureat 40° C. Formed magnetic microparticles were taken out, placed into avessel and allowed to stand at 40° C. for 30 min. The magneticmicroparticles were washed twice with 25 ml of 1.4 wt % aqueous ammonia.Thereafter, the magnetic microparticles were subjected to centrifugationfor 5 min. using a centrifugal separator at a rotation speed of 3,000rpm. The precipitated magnetic microparticles were placed in a samplebottle. Subsequently, after heating at 110° C. for 5 min., 0.14 g ofstearic acid was added and heated at 110° C., and stirring and beingallowed to stand were repeated for 15 min. Then, 10 ml of degassed waterwas added and allowed to stand at 14° C. overnight and subjected todialysis using pure water to obtain magnetic microparticles to whichstearic acid was chemically bonded. The particle size (r) was determinedsimilarly to the foregoing formation method 1.

TABLE 1 Magnetic Micro- Organic Organic Organic Micro- particle Com-Com- Com- particle Formation pound pound pound Particle Size No. MethodA B C r (nm) r1 (nm) 1 1 A-1 — — 15 2 1 A-2 — — 15 3 1 A-3 — — 15 4 1A-3 — — 20 5 1 A-4 — — 15 6 1 A-5 — — 15 7 1 A-6 — — 5 8 1 A-6 — — 10 91 A-6 — — 20 10 1 A-6 — — 30 11 2 A-2 — — 15 12 12 2 A-4 — — 15 12 13 2A-6 — — 1 0.8 14 2 A-6 — — 2.5 2 15 2 A-6 — — 5 4 16 2 A-6 — — 10 8 17 2A-6 — — 20 16 18 3 — B-1 C-2 10 19 3 — B-4 C-2 10 20 3 — B-5 C-2 10 21 4— B-1 C-1 10 8 22 4 — B-4 C-1 10 8 23 4 — B-5 C-1 10 8 24 4 — B-1 C-2 108 25 4 — B-2 C-2 10 8 26 4 — B-3 C-2 10 8 27 4 — B-4 C-2 10 8 28 4 — B-5C-2 10 8 29 1 — — — 10 30 1 — — — 20 31 5 — — — 10 32 5 — — — 20

Examples 1-44 and Comparative Examples 1-10

Manufacturing Method 1

Using magnetic microparticles, as shown in Table 2, magnetic vesicularparticles were prepared in the following manner. In a 10 ml eggplanttype flask, 1.5 mg of dipalmitoylphosphatidylcholine (DPPC), 8.5 mg ofphosphatidylethanolamine and 20 mg of phosphatidylcholine were dissolvedin 2.0 ml of chloroform to form a homogeneous solution. Thereafter,solvents in the solution were removed under reduced pressure and driedovernight in a desiccator to form a DPPC film in the flask.

Subsequently, distilled water was added to the magnetic microparticlesdescribed in Table 2 to form a slurry suspension containing the magneticmicroparticles at a concentration of 10 μg/μl. The slurry suspension wasadded into the flask, at 0.5-6.0 μm and further thereto was added 0.4 mlof a phosphoric acid-buffered physiological saline of a 10-foldconcentration was added. The thus formed mixture was stirred byrepeating ultrasonic stirring over 60 sec. and then pausing over 30 sec.for a period of 60 min. After completion of ultrasonic stirring, themixture was subjected to centrifugal separation for 15 min. using acentrifugal separator at a rotation speed of 3,300 rpm. The obtainedsupernatant was further subjected to centrifugation at a rotation speedof 7,500 rpm for 50 min., whereby lipid membrane-magnetic vesicularparticles having a grain size (R) shown in Table 2 were obtained asprecipitates. The grain size of the magnetic vesicular particles wasobserved using a transmission electron microscope. The grain size (R) isan averaged value of 20 magnetic vesicular particles.

Manufacturing Method 2

In a 10 ml eggplant type flask, dimethyloctadecylammonium bromide,dioleylphosphatidylcholine (DOPE) and C-2 ceramide at a ratio of 1:1:2(in a total amount of 2 ml) were dissolved in 2.0 ml of chloroform toform a homogeneous solution. Thereafter, solvents of the solution wereremoved under reduced pressure and dried overnight in a desiccator toform a DPPC film in the flask.

Subsequently, distilled water was added to the magnetic microparticlesdescribed in Table 2 to form a slurry suspension containing magneticmicroparticles at a concentration of 10 μg/μl. The slurry suspension wasadded into the flask, within an amount of 0.5 to 6.0 μm and furtherthereto, a phosphoric acid-buffered physiological saline at a 10-foldconcentration was added in an amount of 1/10 of the volume of the slurrysuspension of magnetic microparticles. The thus formed mixture wasstirred by repeating ultrasonic stirring over 60 sec. and pausing over30 sec. for a period of 60 min. After completion of ultrasonic stirring,the mixture was subjected to centrifugal separation for 15 min. using acentrifugal separator at a rotation speed of 3,300 rpm. The obtainedsupernatant was further subjected to centrifugation at a rotation speedof 7,500 rpm for 50 min., whereby phospholipid membrane-magneticvesicular particles having a grain size (R) shown in Table 2 wereobtained as precipitates. The grain size (R) of the phospholipidmembrane-magnetic vesicular particles was determined similarly to theforegoing manufacturing method of the lipid membrane-magnetic vesicularparticles.

Manufacturing Method 3

A mixture of 40 mg of DPPC, 1.2 mg of a block copolymer ofpolyethyleneoxide and polypropyleneoxide (pluronic F-88, produced byADEKA Co., Ltd.) and 900 mg of ethanol was placed into a stainless steelautoclave and the interior of the autoclave was heated to 60° C., then,13 g of liquid carbon dioxide was added thereto. The pressure within theautoclave was increased from 50 kg/cm² to 200 kg/cm² and DPPC wasallowed to be dissolved in the supercritical carbon dioxide, whilestirring within the autoclave. Subsequently, physiological saline wasadded to the magnetic microparticles described in Table 2 to form aslurry suspension containing magnetic microparticles at a concentrationof 10 μg/μl.

While stirring the supercritical carbon dioxide solution, the foregoingslurry suspension was continuously added thereto within a range of 0.5to 6.0 ml. The interior of the autoclave was then evacuated to dischargethe carbon dioxide.

The mixture was subjected to centrifugal separation for 15 min. using acentrifugal separator at a rotation speed of 3,300 rpm. The obtainedsupernatant was further subjected to centrifugation at a rotation speedof 7,500 rpm for 50 min., whereby phospholipid membrane-magneticvesicular particles having a grain size (R) shown in Table 2 wereobtained as precipitates. The grain size (R) of the phospholipidmembrane-magnetic vesicular particles was determined similarly to theforegoing manufacturing method of the lipid membrane-magnetic vesicularparticles.

Manufacturing Method 4

Dimethyloctadecylammonium bromide, dioleylphosphatidylcholine (DOPE) andC-2 ceramide were mixed at a ratio of 1:1:2 (in a total amount of 2 ml).The obtained mixture and 900 mg of ethanol was placed into a stainlesssteel autoclave and the interior of the autoclave was heated to 60° C.,then, 13 g of liquid carbon dioxide was added thereto. The pressurewithin the autoclave was increased from 50 kg/cm² to 200 kg/cm² and DOPEwas allowed to be dissolved in the supercritical carbon dioxide, whilestirring within the autoclave. Subsequently, physiological saline wasadded to the magnetic microparticles described in Table 2 to form aslurry suspension containing magnetic microparticles at a concentrationof 10 μg/μl.

While stirring supercritical carbon dioxide solution, the foregoingslurry suspension was continuously added thereto within the range of 0.5to 6.0 ml. The interior of the autoclave was evacuated to dischargecarbon dioxide. The mixture was subjected to centrifugal separation for15 min. using a centrifugal separator at a rotation speed of 3,300 rpm.The obtained supernatant was further subjected to centrifugation at arotation speed of 7,500 rpm for 50 min., whereby phospholipidmembrane-magnetic vesicular particles having a grain size (R) shown inTable 2 were obtained as precipitates. The grain size (R) of thephospholipid membrane-magnetic vesicular particles was determinedsimilarly to the foregoing manufacturing method of the lipidmembrane-magnetic vesicular particles.

Manufacturing Method 5

Lipid having a maleimide group (EMC-DPPE) was prepared in accordancewith Example 1 of JP-A No. 11-106391. Thus, 1.5 mg of the EMC-DPPE, 8.5mg of phosphatidylethanolamine, 20 mg of phosphatidylcholine and 900 mlof ethanol were placed into a stainless steel autoclave and the interiorof the autoclave was heated to 60° C., then, 13 g of liquid carbondioxide was added thereto. The pressure in the interior of the autoclavewas increased from 50 kg/cm² to 200 kg/cm² and lipid was allowed to bedissolved in the supercritical carbon dioxide, while stirring within theautoclave. Subsequently, physiological saline was added to the magneticmicroparticles described in Table 2 to form a slurry suspensioncontaining magnetic microparticles at a concentration of 10 μg/μl.

While stirring the supercritical carbon dioxide solution, the foregoingslurry suspension was continuously added thereto at 0.5-6.0 ml. Theinterior of the autoclave was evacuated to discharge the carbon dioxide.

To this solution, monoclonal antibody G-22 was attached in accordancewith the method described in Example 1 of JP-A No. 11-106391. Themixture was subjected to centrifugal separation for 15 min. using acentrifugal separator at a rotation speed of 3,300 rpm. The obtainedsupernatant was further subjected to centrifugation at a rotation speedof 7,500 rpm for 50 min., whereby phospholipid membrane-magneticvesicular particles having a grain size (R) shown in Table 2 wereobtained as precipitates. The grain size (R) of the phospholipidmembrane-magnetic vesicular particles was determined similarly to theforegoing manufacturing method of the lipid membrane-magnetic vesicularparticles.

TABLE 2 Magnetic Manu- Par- Size Vesicular fac- Magnetic ticle ParticleRatio Particle turing Micro- Size r Size R/(r × No. Method particle (nm)R (nm) 100) Example 1 1 1 1 15 120 0.080 Example 2 2 1 2 15 120 0.080Example 3 3 1 3 15 120 0.080 Example 4 4 1 4 20 120 0.060 Example 5 5 15 15 120 0.080 Example 6 6 1 6 15 120 0.080 Example 7 7 1 7 5 100 0.200Example 8 8 1 7 5 150 0.300 Example 9 9 1 8 10 100 0.100 Example 10 10 19 20 100 0.050 Example 11 11 1 10 30 150 0.050 Comparative 12 1 10 30100 0.033 Example 1 Example 12 13 2 7 5 100 0.200 Example 13 14 2 8 10100 0.100 Example 14 15 2 9 20 150 0.075 Example 15 16 2 10 30 150 0.050Example 16 17 2 11 15 120 0.080 Example 17 18 2 12 15 120 0.080Comparative 19 2 13 1 180 1.800 Example 2 Example 18 20 2 13 1 150 1.500Example 19 21 2 14 2.5 150 0.600 Example 20 22 2 15 5 100 0.200 Example21 23 2 16 10 100 0.100 Example 22 24 2 17 20 100 0.050 Example 23 25 218 10 100 0.100 Example 24 26 2 19 10 100 0.100 Example 25 27 2 20 10100 0.100 Example 26 28 3 11 10 100 0.100 Example 27 29 3 12 10 1000.100 Example 28 30 3 13 10 100 0.100 Example 29 31 4 5 15 100 0.067Example 30 32 4 8 10 100 0.100 Example 31 33 4 16 10 100 0.100 Example32 34 4 18 10 100 0.100 Example 33 35 4 19 10 100 0.100 Example 34 36 420 10 100 0.100 Example 35 37 4 24 10 100 0.100 Example 36 38 4 25 10100 0.100 Example 37 39 4 27 10 100 0.100 Example 38 40 4 28 10 1000.100 Example 39 41 5 5 15 100 0.067 Example 40 42 5 8 10 100 0.100Example 41 43 5 16 10 100 0.100 Example 42 44 5 18 10 100 0.100 Example43 45 5 24 10 100 0.100 Example 44 46 5 25 10 100 0.100 Comparative 47 129 15 120 0.080 Example 3 Comparative 48 1 30 20 120 0.060 Example 4Comparative 49 1 31 10 150 0.150 Example 5 Comparative 50 1 32 20 1500.075 Example 6 Comparative 51 2 29 15 120 0.080 Example 7 Comparative52 2 30 20 120 0.060 Example 8 Comparative 53 2 31 10 150 0.150 Example9 Comparative 54 2 32 20 150 0.075 Example 10

Using the lipid membrane-magnetic vesicular particles obtained above,the following tests (Tests 1-6) were conducted.

Test 1

The magnetic vesicular particles shown in Table 3 were each diluted withan isotonic glucose solution to a concentration of 10 mg iron/ml. Thissolution was injected into a vein of rabbits to which experimental tumorVX-2 was transplanted to their liver in different sizes. After theelapse of time shown in Table 3, the rabbits were observed in anultrasonic imaging diagnostic apparatus to judge the size ofdiscriminable liver cancer. Results thereof are shown in Table 3.

As can be seen from Table 3, it was proved that lipid membrane-magneticvesicular particles of this invention had no problem with respect todiscriminable tumor size, as compared to comparative examples andvariation in size of discriminable tumor was less than in thecomparative samples, even after an elapse of time.

TABLE 3 Magnetic Vesicular Size of Discriminable Tumor (mm) ParticleAfter After After After After No. 30 min. 1 hr. 6 hr. 12 hr. 24 hr.Example 1 1 5 5 8 10 10 Example 2 2 5 5 8 10 10 Example 3 3 5 5 8 10 10Example 4 4 5 5 8 10 10 Example 5 5 5 5 8 10 10 Example 6 6 5 5 8 10 10Example 7 7 5 5 8 10 10 Example 8 8 5 5 8 10 10 Example 9 9 5 5 8 10 10Example 10 10 5 5 8 10 10 Example 12 13 5 5 5 8 10 Example 13 14 5 5 5 810 Example 14 15 5 5 5 8 10 Example 15 16 5 5 5 8 10 Example 16 17 5 5 58 10 Example 17 18 5 5 5 8 10 Comparative 19 8 10 10 15 —*¹ Example 2Example 18 20 5 5 5 8 10 Example 19 21 5 5 5 8 10 Example 20 22 5 5 5 810 Example 21 23 5 5 5 8 10 Example 22 24 5 5 5 8 10 Example 23 25 5 5 58 10 Example 24 26 5 5 5 8 10 Example 25 27 5 5 5 8 10 Comparative 47 510 —*¹ —*¹ —*¹ Example 3 Comparative 48 5 8 10 —*¹ —*¹ Example 4Comparative 49 5 8 10 —*¹ —*¹ Example 5 Comparative 50 5 8 10 —*¹ —*¹Example 6 *¹Function as a contrast medium was not noted at all.Test 2

The magnetic vesicular particles shown in Table 4 were each diluted withan isotonic glucose solution to a concentration of 10 mg iron/ml. Thissolution was locally injected into the breast cancer tumor site ofmousses into which a cell line of human breast cancer was hypodermicallytransplanted. After the elapse of time as shown in Table 4, the rabbitswere observed in a nuclear magnetic resonance imaging diagnosticapparatus to determine the size of discriminable tumors. Results thereofare shown in Table 4.

As can be seen from Table 4, it was proved that lipid membrane-magneticvesicular particles of this invention were discriminable even after anelapse of time, as compared to comparative examples.

TABLE 4 Magnetic Vesicular Size of Discriminable Tumor (mm) ParticleAfter After After After After No. 30 min. 1 hr. 6 hr. 12 hr. 24 hr.Example 21 23 5 5 5 8 10 Example 22 24 5 5 5 8 10 Example 23 25 5 5 5 810 Example 24 26 5 5 5 8 10 Example 25 27 5 5 5 8 8 Example 26 28 5 5 58 8 Example 27 29 5 5 5 8 8 Example 28 30 5 5 5 8 8 Example 29 31 5 5 55 8 Example 30 32 5 5 5 5 8 Example 31 33 5 5 5 5 8 Example 32 34 5 5 55 8 Example 33 35 5 5 5 5 8 Example 34 36 5 5 5 5 8 Example 35 37 5 5 55 8 Example 36 38 5 5 5 5 8 Example 37 39 5 5 5 5 8 Example 38 40 5 5 55 8 Comparative 51 5 8 10 15 —*¹ Example 7 Comparative 52 5 8 10 15 —*¹Example 8 Comparative 53 5 5 8 10 15 Example 9 Comparative 54 5 5 8 1015 Example 10 *¹Function as a contrast medium was not noted at all.Test 3

The magnetic vesicular particles shown in Table 5 were each diluted withan isotonic glucose solution to a concentration of 10 mg iron/ml. Thissolution was injected into a vein of rats into which a cell line ofhuman malignant glioma had been transplanted. After an elapse of time asshown in Table 5, the rats were observed in a nuclear magnetic resonanceimaging diagnostic apparatus to determine the size of discriminabletumors. Results thereof are shown in Table 5.

TABLE 5 Magnetic Vesicular Size of Discriminable Tumor (mm) ParticleAfter After After After After After No. 30 min. 1 hr. 6 hr. 12 hr. 24hr. 36 hr. Example 13 14 5 5 5 8 10 10 Example 23 25 5 5 5 8 10 10Example 29 31 5 5 5 5 8 10 Example 30 32 5 5 5 5 8 10 Example 31 33 5 55 5 8 10 Example 32 34 5 5 5 5 8 10 Example 35 37 5 5 5 5 8 10 Example36 38 5 5 5 5 8 10 Example 39 41 5 5 5 5 5 8 Example 40 42 5 5 5 5 5 8Example 41 43 5 5 5 5 5 5 Example 42 44 5 5 5 5 5 8 Example 43 45 5 5 55 5 5 Example 44 46 5 5 5 5 5 5 Comparative 51 5 5 8 10 —*¹ —*¹ Example7 Comparative 52 5 5 8 10 —*¹ —*¹ Example 8 Comparative 53 5 5 8 8 10—*¹ Example 9 Comparative 54 5 5 8 8 10 —*¹ Example 10 *¹Function as acontrast medium was not noted at all.Test 4

The magnetic vesicular particles shown in Table 6 were each diluted withan isotonic glucose solution to a concentration of 10 mg iron/ml. Thissolution was locally injected into the breast cancer site of moussesinto which a cell line of low molecular type line cancer, originatedfrom human lung was transplanted immediately below the pleura. After 30min. or 24 hr., exposure to an alternating magnetic field was conductedunder the following conditions:

frequency: 375 Hz, coil to generate a magnetic field: 300 mm indiameter, magnetic field intensity: 6 mT, current: 225 A, output: 3 KW,and distance from an applicator: 20 mm.

A fiberoptic thermometer was arranged in and near the tumor site todetermine temperatures of tumor and normal sites. The time of the tumorsite reaching 43° C. was determined, at which time the temperature ofthe normal site was also determined. Results thereof are shown in Table6.

As can be seen from Table 6, it was proved that when thermotherapy wasconducted, the lipid membrane-magnetic vesicular particles of thisinvention efficiently raised the temperature of only the tumor sitewithin a short period of time, enabling performance of efficientthermotherapy irrespective of elapse of time after local injection, ascompared to comparative examples.

TABLE 6 Mag- netic Normal Ve- Site Heating (1)*² Heating (2)*³ sicularTem- Tem- Tem- Particle perature*¹ Time*⁴ perature*⁵ Time*⁴ perature*⁵No. (° C.) (min) (° C.) (min) (° C.) Exam- 9 32.8 5.0 34.5 10.5 36.9 ple9 Exam- 10 33.5 6.0 35.2 12.5 37.8 ple 10 Com- 12 32.9 15.0 38.5 25.042.5 parative Exam- ple 1 Exam- 14 33.3 5.0 35.1 10.0 36.9 ple 13 Exam-23 33.8 3.5 34.2 7.5 35.6 ple 21 Exam- 38 33.0 3.5 33.9 6.0 35.1 ple 36Com- 53 33.1 5.0 34.8 20.0 40.5 parative Exam- ple 9 *¹normal sitetemperature before subjected to alternating magnetic field heating*²alternating magnetic field heating at 30 min after injection*³alternating magnetic field heating at 24 hr. after injection *⁴timenecessary to reach 43° C. *⁵normal site temperature (° C.)Test 5

The magnetic vesicular particles shown in Table 7 were each diluted withan isotonic glucose solution to a concentration of 10 mg iron/ml. Thissolution was injected into veins of rats into which a cell line of humanmalignant glioma was transplanted. After 1 hr. or the time shown inTable 7, exposure to an alternating magnetic field was continuouslyconducted under the following conditions:

frequency: 370 Hz, applicator to generate a magnetic field: 310 mm incoil diameter, magnetic field intensity: 8 mT, current: 300 A, output: 5KW, and distance from the applicator: 20 mm.

A fiberoptic thermometer was arranged in the tumor site to determinetemperatures of the tumor and the normal site. The time of the tumorsite reaching 43° C. was determined, at which time the temperature ofthe normal site was also determined. Results thereof are shown in Table7.

TABLE 7 Magnetic Normal Vesicular Site Heating (1)*² Heating (2)*³Heating (3)*⁴ Particle Temperature*¹ Time*⁵ Temperature*⁶ Time*⁵Temperature*⁶ Time*⁵ Temperature*⁶ No. (° C.) (min) (° C.) (min) (° C.)(min) (° C.) Example 13 14 33.5 5.0 34.8 12.0 37.6 25.0 42.4 Example 2325 32.6 5.0 34.4 10.0 36.8 18.0 39.8 Example 29 31 33.1 5.0 34.3 8.536.8 15.0 38.4 Example 30 32 33.0 5.0 35.0 8.5 36.4 15.0 38.9 Example 3133 32.6 4.0 34.5 7.0 35.4 13.0 37.5 Example 35 37 32.8 4.0 34.3 7.0 35.912.0 37.4 Example 36 38 33.5 3.5 34.1 5.5 35.1 13.0 37.9 Example 39 4133.4 3.5 34.4 5.0 34.9 10.0 36.5 Example 40 42 33.1 3.5 33.8 5.0 34.710.0 36.3 Example 41 43 32.9 3.0 33.5 4.0 34.6 7.0 35.5 Example 43 4533.0 3.0 33.9 3.5 34.1 5.0 34.8 Example 44 46 33.4 3.0 33.7 3.5 34.3 5.034.6 Comparative 53 33.2 5.0 34.8 —*⁷ —*⁷ —*⁷ —*⁷ Example 9 *¹normalsite temperature before subjected to alternating magnetic field heating*²alternating magnetic field heating at 1 hr. after injection*³alternating magnetic field heating at 6 hr. after injection*⁴alternating magnetic field heating at 24 hr. after injection *⁵timenecessary to reach 43° C. *⁶normal site temperature (° C.) *⁷notemperature increase even after subjected to alternating magnetic fieldheating for 30 min.

As can be seen from Table 7, it was proved that when thermotherapy wasconducted, the lipid membrane-magnetic vesicular particles of thisinvention efficiently raised a temperature of only a tumor site within ashort period of time, as compared to the comparative examples. It wasfurther shown that enhanced accumulation at the tumor site enablesperformance of efficient therapy without newly supplying magnetic grainswhen applying therapy a few times.

Test 6

The magnetic vesicular particles shown in Table 8 were each diluted withan isotonic glucose solution to a concentration of 10 mg iron/ml. Thissolution was injected through intravenous injection to mousses intowhich a cell line of human breast cancer was hypodermicallytransplanted. After 2 hr. and 24 hr., exposure to an alternatingmagnetic field was conducted similarly to the foregoing test 5. After 6days since the first intravenous injection, a second intravenousinjection was conducted, and again after 2 hr. and 24 hr., exposure toan alternating magnetic field was conducted similarly to the foregoingtest 5. Again, after 15 days since the second intravenous injection, athird intravenous injection was conducted.

After 1 hr. since the respective first, second and third injections ofthe magnetic vesicular particles, observation was conducted in a nuclearmagnetic resonance imaging diagnostic apparatus to determine the size ofa glioma tumor site from a diagnosis image. Results thereof are shown inTable 8. In any case of the foregoing, capability of achieving contrastas a contrast medium for tumor was at a level acceptable in practice.

TABLE 8 Magnetic Vesicular Particle Size of Tumor (mm) No. 1st (m) 2nd(mm) 3rd (mm) Example 21 23 12 8 —*¹ Example 23 25 11 8 —*¹ Example 3133 10 7 —*¹ Example 32 34 12 7 —*¹ Example 35 37 11 7 —*¹ Example 36 3811 7 —*¹ Example 41 43 10 5 —*¹ Example 42 44 12 6 —*¹ Example 43 45 105 —*¹ Example 44 46 12 5 —*¹ Comparative 53 11 9 6 Example 9 *¹no tumorwas noted in nuclear magnetic resonance imaging diagnosis

As can be seen from Table 8, it was proved that magnetic vesicularparticles of this invention can not only provide both functions as animaging agent (or contrast medium) and a therapeutic agent but alsoefficiently function as a therapeutic agent.

1. A method of manufacturing a preparation containing magnetic vesicularparticles, the method comprising: (a) mixing at least one lipid membraneconstituent and a supercritical carbon dioxide to form a mixture, (b)adding to the mixture a dispersion containing magnetic nanoparticles towhich an organic compound is bonded, and (c) discharging the carbondioxide to form the magnetic vesicular particles including the magneticnanoparticles within a lipid membrane, wherein the magnetic vesicularparticles, each includes at least one magnetic nanoparticle within alipid membrane, an organic compound having at least two groups selectedfrom the group consisting of a hydroxyl group, a carboxyl group, acarbamoyl group, an amino group, a mercapto group, a sulfo group, adithio group, a thiocarboxyl group and a dithiocarboxyl group is bondedto the magnetic nanoparticle, and the magnetic vesicular particlessatisfy the following equation:0.05≦R/(r×100)≦1.5 wherein R represents an average grain size of themagnetic vesicular particles and r represents an average particle sizeof magnetic nanoparticles included in the magnetic vesicular particleswherein the organic compound is a compound selected from the groupconsisting of phthalic acid, isophthalic acid, therephthalic acid,succinic acid, glutaric acid, adipic acid, pimelic acid, fumaric acid,maleic acid, 2-mercaptoamine, 6-aminehexanethiol, 2-mercaptopropionicacid, asparagic acid, glutamine, malic acid, oxaloacetic acid,2-ketoglutaric acid, serine, threonine, cysteine, cysteic acid, cystine,N-acetylcysteine, cysteine ethyl ester, and dithiothreitol.
 2. Themethod of claim 1, wherein the magnetic vesicular particles satisfy thefollowing equation:0.05≦R/(r×100)≦1.0 wherein R and r are the same as defined in claim 1.3. The method of claim 1, wherein the magnetic nanoparticles have anaverage particle size of 1 to 30 nm and are comprised of a ferrite. 4.The method of claim 1, wherein the magnetic vesicular particles areliposomes formed of the lipid membrane and including at least onemagnetic nanoparticle within the lipid membrane.
 5. The method of claim4, wherein the liposomes exhibit a positive surface charge.
 6. Themethod of claim 1, wherein the preparation further comprises an imagingagent or a therapeutic agent for a tumor.
 7. The method of claim 6,wherein the therapeutic agent for a tumor is bonded directly or througha linking material to the lipid membrane.
 8. The method of claim 6,wherein the imaging agent is a contrast medium for use in ultrasonicimaging diagnosis, nuclear magnetic resonance imaging diagnosis or X-rayimaging diagnosis.